Tango Biosciences, 2201 W. Campbell Park Drive, Chicago, IL 60612-4092 USA.
Protein Eng Des Sel. 2024 Jan 29;37. doi: 10.1093/protein/gzae014.
Antibodies play a crucial role in monitoring post-translational modifications, like phosphorylation, which regulates protein activity and location; however, commercial polyclonal and monoclonal antibodies have limitations in renewability and engineering compared to recombinant affinity reagents. A scaffold based on the Forkhead-associated domain (FHA) has potential as a selective affinity reagent for this post-translational modification. Engineered FHA domains, termed phosphothreonine-binding domains (pTBDs), with limited cross-reactivity were isolated from an M13 bacteriophage display library by affinity selection with phosphopeptides corresponding to human mTOR, Chk2, 53BP1, and Akt1 proteins. To determine the specificity of the representative pTBDs, we focused on binders to the pT543 phosphopeptide (536-IDEDGENpTQIEDTEP-551) of the DNA repair protein 53BP1. ELISA and western blot experiments have demonstrated the pTBDs are specific to phosphothreonine, demonstrating the potential utility of pTBDs for monitoring the phosphorylation of specific threonine residues in clinically relevant human proteins.
抗体在监测翻译后修饰（如磷酸化）方面发挥着至关重要的作用，磷酸化调节蛋白质的活性和位置；然而，与重组亲和试剂相比，商业的多克隆和单克隆抗体在可更新性和工程方面存在局限性。基于 Forkhead 相关结构域（FHA）的支架具有作为这种翻译后修饰的选择性亲和试剂的潜力。通过使用与人类 mTOR、Chk2、53BP1 和 Akt1 蛋白的磷酸肽相对应的亲和选择，从 M13 噬菌体展示文库中分离出具有有限交叉反应性的工程 FHA 结构域，称为磷酸苏氨酸结合结构域（pTBD）。为了确定代表性 pTBD 的特异性，我们专注于与 DNA 修复蛋白 53BP1 的 pT543 磷酸肽（536-IDEDGENpTQIEDTEP-551）结合的结合物。ELISA 和 Western blot 实验表明，pTBD 特异性识别磷酸苏氨酸，这表明 pTBD 具有监测临床相关人类蛋白质中特定苏氨酸残基磷酸化的潜在用途。
